ab174447 – Clusterin (Apolipoprotein J) Human SimpleStep ELISA™ Kit

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ab174447 – Clusterin

(Apolipoprotein J)

Human

SimpleStep ELISA™ Kit

Instructions for Use

For the quantitative measurement of Clusterin (Apolipoprotein J) in human plasma, serum and cell culture supernatants.

This product is for research use only and is not intended for diagnostic use.

Version 1 Last Updated 3 March 2015

Table of Contents

INTRODUCTION

1.

2.

BACKGROUND

ASSAY SUMMARY

GENERAL INFORMATION

6.

7.

8.

3.

4.

5.

PRECAUTIONS

STORAGE AND STABILITY

MATERIALS SUPPLIED

MATERIALS REQUIRED, NOT SUPPLIED

LIMITATIONS

TECHNICAL HINTS

ASSAY PREPARATION

9.

REAGENT PREPARATION

10. STANDARD PREPARATION

11. SAMPLE PREPARATION

12. PLATE PREPARATION

ASSAY PROCEDURE

13. ASSAY PROCEDURE

DATA ANALYSIS

14. CALCULATIONS

15. TYPICAL DATA

16. TYPICAL SAMPLE VALUES

17. SPECIES REACTIVITY

RESOURCES

18. TROUBLESHOOTING

19. NOTES

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5

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1

INTRODUCTION

1.

BACKGROUND

Abcam’s Clusterin (Apolipoprotein J)

in vitro

SimpleStep ELISA™

(Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Clusterin (Apolipoprotein J) protein in human plasma, serum and cell culture supernatants.

The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex

(capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

Clusterin (CLU) is composed of an antiparallel disulfide-linked heterodimer of an alpha chain and a beta chain that self-associate and can form higher oligomers. It is ubiquitously expressed in various tissues. Clusterin functions as an extracellular chaperone that prevents aggregation of nonnative proteins. It does not require ATP and does not refold proteins by itself. Clusterin does so by maintaining partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones, such as HSPA8/HSC70. Clusterin prevents stress-induced aggregation of blood plasma proteins by inhibiting the formation of amyloid fibrils through APP, APOC2, B2M,

CALCA, CSN3, SNCA and aggregation-prone LYZ variants (in vitro). It can bind to cell surface receptors and trigger internalization of the

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2

INTRODUCTION

chaperone-client complex and subsequent lysosomal or proteasomal degradation.

Secreted isoforms protect cells against apoptosis and against cytolysis by complement. Intracellular isoforms interact with ubiquitin and SCF

(SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes and promote the ubiquitination and subsequent proteasomal degradation of target proteins. Clusterin can retrotranslocate from the secretory compartments to the cytosol upon induction of stress. Nuclear isoforms promote apoptosis. Mitochondrial isoforms suppress BAXdependent release of cytochrome c into the cytoplasm and inhibit apoptosis.

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3

INTRODUCTION

2.

ASSAY SUMMARY

Remove appropriate number of antibody coated well strips.

Equilibrate all reagents to room temperature. Prepare all reagents, samples, standards as instructed.

and

Add standard or sample to appropriate wells.

Add Antibody Cocktail to all wells. Incubate temperature. at room

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Aspirate and wash each well.

Add TMB Substrate to each well and incubate. Add Stop Solution at a defined

Alternatively, endpoint. record color development kinetically after

TMB substrate addition.

4

GENERAL INFORMATION

3.

PRECAUTIONS

Please read these instructions carefully prior to beginning the assay.

All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance.

4.

STORAGE AND STABILITY

Store kit at 2-8ºC immediately upon receipt.

Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in sections 9 & 10.

5.

MATERIALS SUPPLIED

Item

10X Human Clusterin Capture Antibody

10X Human Clusterin Detector Antibody

Human Clusterin Lyophilized Purified Protein

Antibody Diluent CPI

10X Wash Buffer PT

5X Cell Extraction Buffer PTR*

50X Cell Extraction Enhancer Solution*

Amount

2 x 300 µL

2 x 300 µL

2 Vials

2 x 3 mL

20 mL

10 mL

1 mL

Storage

Condition

(Before

Preparation)

+2-8ºC

+2-8ºC

+2-8ºC

+2-8ºC

+2-8ºC

+2-8ºC

+2-8ºC

TMB Substrate

Stop Solution

Sample Diluent NS

Pre-Coated 96-Well Microplate (12 x 8 wells)

Plate Seal

12 mL

12 mL

12 mL

96 Wells

1

+2-8ºC

+2-8ºC

+2-8ºC

+2-8ºC

+2-8ºC

*Cell and tissue extracts have not been tested with this kit but for your convenience we have provided 5X Cell Extraction Buffer PTR and 50X Cell

Extraction Enhancer Solution.

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GENERAL INFORMATION

6.

MATERIALS REQUIRED, NOT SUPPLIED

These materials are not included in the kit, but will be required to successfully utilize this assay:

Microplate reader capable of measuring absorbance at 450 or

600 nm.

Method for determining protein concentration (BCA assay recommended).

Deionized water.

PBS (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl,

2.7 mM KCl, pH 7.4).

Multi- and single-channel pipettes.

Tubes for standard dilution.

Plate shaker for all incubation steps.

Optional: Phenylmethylsulfonyl Fluoride (PMSF) (or other protease inhibitors).

7.

LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic procedures.

Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted.

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GENERAL INFORMATION

8.

TECHNICAL HINTS

Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers.

Avoid foaming or bubbles when mixing or reconstituting components.

Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions.

Ensure plates are properly sealed or covered during incubation steps.

Complete removal of all solutions and buffers during wash steps is necessary to minimize background.

As a guide, typical ranges of sample concentration for commonly used sample types are shown below in Sample Preparation

(section 11).

All samples should be mixed thoroughly and gently.

Avoid multiply freeze/thaw of samples.

Incubate ELISA plates on a plate shaker during all incubation steps.

When generating positive control samples, it is advisable to change pipette tips after each step.

The provided 5X Cell Extraction Buffer contains phosphatase inhibitors. Protease inhibitors can be added if required.

The provided 50X Cell Extraction Enhancer Solution may precipitate when stored at + 4ºC. To dissolve, warm briefly at

+ 37ºC and mix gently. The 50X Cell Extraction Enhancer Solution can be stored at room temperature to avoid precipitation.

This kit is sold based on number of tests. A ‘test’ simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product.

Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions.

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ASSAY PREPARATION

9.

REAGENT PREPARATION

Equilibrate all reagents to room temperature (18-25°C) prior to use. The kit contains enough reagents for 96 wells.

The sample volumes below are sufficient for 48 wells (6 x 8-well strips); adjust volumes as needed for the number of strips in your experiment.

Prepare only as much reagent as is needed on the day of the experiment. Capture and Detector Antibodies have only been tested for stability in the provided 10X formulations.

9.1

9.2

9.3

1X Wash Buffer PT

Prepare 1X Wash Buffer PT by diluting 10X Wash Buffer PT with deionized water. To make 50 mL 1X Wash Buffer PT combine 5 mL 10X Wash Buffer PT with 45 mL deionized water. Mix thoroughly and gently.

Antibody Cocktail

Prepare Antibody Cocktail by diluting in Antibody Diluent

CPI. To make 3 mL of the Antibody Cocktail combine

300 µL 10X Capture Antibody and 300 µL 10X Detector

Antibody with 2.4 mL Antibody Diluent CPI. Mix thoroughly and gently.

1X Cell Extraction Buffer PTR

(For cell & tissue extracts only)

If required, prepare 1X Cell Extraction Buffer PTR by diluting

5X Cell Extraction Buffer PTR and 50X Cell Extraction

Enhancer Solution to 1X with deionized water. To make

10 mL 1X Cell Extraction Buffer PTR combine 7.8 mL deionized water, 2 mL 5X Cell Extraction Buffer PTR and

200 µL 50X Cell Extraction Enhancer Solution Mix thoroughly and gently.

Alternative

– Enhancer may be added to Cell Extraction

Buffer after extraction of cells or tissue. Refer to note in

Troubleshooting section.

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ASSAY PREPARATION

10.

STANDARD PREPARATION

Prepare serially diluted standards immediately prior to use. Always prepare a fresh set of positive controls for every use.

The following table describes the preparation of a standard curve for duplicate measurements (recommended).

10.1 Reconstitute the Clusterin human standard by adding 250 µL water by pipette. Mix thoroughly and gently. Hold at room temperature for 3 minutes and mix gently. This is the

400 ng/mL

Standard #1

Solution

10.2 Label eight tubes, Standards 2– 8 and add 125

Sample Diluent NS into each tube.

μL of

10.3 Use the Stock Standard to prepare the following dilution series. Standard #8 contains no protein and is the Blank control:

125 µL

µ

125 µL

µ

125 µL

µ

125 µL

µ

125 µL

µ

125 µL

µ

400 ng/mL

200 ng/mL

100 ng/mL

50 ng/mL

25 ng/mL

12.5

ng/mL

6.25

ng/mL

0 ng/mL

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ASSAY PREPARATION

11.

SAMPLE PREPARATION

TYPICAL SAMPLE DYNAMIC RANGE

Sample Type Range

Human Serum

Human Plasma - EDTA

Human Plasma - Heparin

HeLa Conditioned Media

1:32,000 – 1:500

1:32,000 – 1:400

1:32,000 – 1:500

1:100 – Neat

11.1.

11.2.

11.3.

Plasma

Collect plasma using citrate, EDTA or heparin. Centrifuge samples at 2,000 x g for 10 minutes. Dilute samples into

Sample Diluent NS and assay. Store un-diluted plasma samples at -20ºC or below for up to 3 months. Avoid repeated freeze-thaw cycles.

Serum

Samples should be collected into a serum separator tube.

After clot formation, centrifuge samples at 2,000 x g for 10 minutes and collect serum. Dilute samples into Sample

Diluent NS and assay. Store un-diluted serum at -20ºC or below. Avoid repeated freeze-thaw cycles.

Cell Culture Supernatants

Centrifuge cell culture media at 2,000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles.

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ASSAY PREPARATION

12.

PLATE PREPARATION

The 96 well plate strips included with this kit are supplied ready to use. It is not necessary to rinse the plate prior to adding reagents.

Unused plate strips should be immediately returned to the foil pouch containing the desiccant pack, resealed and stored at 4°C.

For each assay performed, a minimum of two wells must be used as the zero control.

For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates).

Differences in well absorbance or “edge effects” have not been observed with this assay.

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ASSAY PROCEDURE

13.

ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room temperature prior to use.

It is recommended to assay all standards, controls and samples in duplicate.

13.1 Prepare all reagents, working standards, and samples as directed in the previous sections.

13.2 Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, reseal and return to 4ºC storage.

13.3 Add 50 µL of each sample and standards to appropriate wells.

13.4 Add 50 µL of the Antibody Cocktail to each well.

13.5 Seal or cover plate and incubate for 1 hour at room temperature on a plate shaker set to 400 rpm.

13.6 Wash each well with 3 x 350 µL 1X Wash Buffer PT. Wash by aspirating or decanting from wells then dispensing 350 µL

1X Wash Buffer PT into each well. Complete removal of liquid at each step is essential for good performance. After the last wash invert the plate and blot it against clean paper towels to remove excess liquid.

13.7 Add 100 µL of TMB Substrate to each well and incubate for

10 minutes in the dark on a plate shaker set to 400 rpm.

13.8 Add 100 µL of Stop Solution to each well. Shake plate on a plate shaker for 1 minute to mix. Record the OD at 450 nm.

This is an endpoint reading.

Alternative to 13.7 – 13.8: Instead of the endpoint reading at

450 nm, record the development of TMB Substrate kinetically. Immediately after addition of TMB Development

Solution begin recording the blue color development with elapsed time in the microplate reader prepared with the following settings:

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ASSAY PROCEDURE

Mode:

Wavelength:

Time:

Kinetic

600 nm up to 15 min

Interval: 20 sec - 1 min

Shaking: Shake between readings

Note that an endpoint reading can also be recorded at the completion of the kinetic read by adding 100 µL Stop

Solution to each well and recording the OD at 450nm.

13.9 Analyze the data as described below.

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DATA ANALYSIS

14.

CALCULATIONS

Subtract average zero standard from all readings. Average the duplicate readings of the positive control dilutions and plot against their concentrations. Draw the best smooth curve through these points to construct a standard curve. Most plate reader software or graphing software can plot these values and curve fit. A four parameter algorithm (4PL) usually provides the best fit, though other equations can be examined to see which provides the most accurate (e.g. linear, semi-log, log/log, 4 parameter logistic). Interpolate protein concentrations for unknown samples from the standard curve plotted.

Samples producing signals greater than that of the highest standard should be further diluted and reanalyzed, then multiplying the concentration found by the appropriate dilution factor.

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DATA ANALYSIS

15.

TYPICAL DATA

TYPICAL STANDARD CURVE

– Data provided for

demonstration purposes only

. A new standard curve must be generated for each assay performed.

Conc.

(ng/mL)

0

6.25

12.5

25

50

100

200

400

Standard Curve Measurements

1

O.D. 450 nm

2

Mean

O.D.

0.080

0.102

0.130

0.172

0.265

0.441

0.699

1.039

0.076

0.110

0.141

0.202

0.318

0.494

0.734

1.051

0.078

0.106

0.135

0.187

0.291

0.468

0.716

1.045

Figure 1.

Example of Clusterin standard curve in Sample Diluent NS. The

Clusterin standard curve was prepared as described in Section 10. Raw data values are shown in the table. Background-subtracted data values (mean +/-

SD) are graphed.

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DATA ANALYSIS

16.

TYPICAL SAMPLE VALUES

SENSITIVITY –

The calculated minimal detectable (MDD) dose is 1.78 ng/mL. The

MDD was determined by calculating the mean of zero standard replicates (n=25) and adding 2 standard deviations then interpolating the corresponding concentrations.

RECOVERY –

(Sample spiking in representative sample matrices)

Sample Type

50% Cell Culture Media

10% Fetal Bovine Serum

10% Fetal Bovine Plasma (sodium citrate)

Average %

Recovery

109 - 111

102 - 103

80 - 81

Range %

110

103

81

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DATA ANALYSIS

LINEARITY OF DILUTION –

Linearity of dilution is determined based on interpolated values from the standard curve. Linearity of dilution defines a sample concentration interval in which interpolated target concentrations are directly proportional to sample dilution.

Recombinant Clusterin was spiked into the following biological samples and diluted in a 2-fold dilution series in Sample Diluent NS. undiluted

1:2

1:4

1:8

1:16

Clusterin (ng/mL)

Average %

Clusterin (ng/mL)

Average %

Clusterin (ng/mL)

Average %

Clusterin (ng/mL)

Average %

Clusterin (ng/mL)

Average %

Sample

Diluent

Conditioned

Media

392.37

100

198.11

101

95.06

97

44.43

91

19.98

81

429.17

100

221.50

103

107.67

100

48.55

91

22.12

82

Fetal

Bovine

Serum

401.79

100

210.97

105

102.90

102

48.80

97

22.85

91

Fetal

Bovine

Plasma

(Sodium

Citrate)

318.11

100

177.16

111

91.82

115

44.61

112

21.70

109

PRECISION –

Mean coefficient of variations of interpolated values from 3 concentrations of normal human serum (pooled donor) within the working range of the assay. n=

CV (%)

Intra-

Assay

9

4.4

Inter-

Assay

3

6.2

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DATA ANALYSIS

Figure 2.

Titration of normal Human serum (pooled donor) within the working range of the assay. Background subtracted data from triplicate measurements are plotted.

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DATA ANALYSIS

Figure 3.

Observed Clusterin concentration in individual donor normal human serum samples (n=10). Mean human Clusterin is 63 µg/mL and values fall within expected normal reference ranges.

Figure 4.

HeLa cells (1x10 6 cells/mL) were cultured in RPMI media supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. Conditioned media was harvested after 48 hours, aliquoted and assayed for endogenous Clusterin levels. Titration of HeLa conditioned media was performed within the working range of the assay.

Background subtracted data from triplicate measurements are plotted. Mean calculated value for endogenous Clusterin was 231 ng/mL.

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DATA ANALYSIS

17.

SPECIES REACTIVITY

This kit detects Clusterin in human plasma, serum and cell culture supernatants.

Human plasma (Sodium Citrate) is not recommended for use in this kit.

Urine and saliva samples have not been tested with this kit.

Please contact our Technical Support team for more information.

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RESOURCES

18.

TROUBLESHOOTING

Problem

Difficulty pipetting lysate; viscous lysate.

Poor standard curve

Low Signal

Large CV

Low sensitivity

Cause

Genomic DNA solubilized

Solution

Prepare 1X Cell Extraction

Buffer PTR (without enhancer). Add enhancer to lysate after extraction.

Check pipettes Inaccurate Pipetting

Improper standard dilution

Incubation times too brief

Prior to opening, briefly spin the stock standard tube and dissolve the powder thoroughly by gentle mixing

Ensure sufficient incubation times; increase to 2 or 3 hour standard/sample incubation

Inadequate reagent volumes or improper dilution

Improper storage of the ELISA kit

Check pipettes and ensure correct preparation

Incubation times with

TMB too brief

Plate is insufficiently washed

Ensure sufficient incubation time until blue color develops prior addition of Stop solution

Review manual for proper wash technique. If using a plate washer, check all ports for obstructions.

Contaminated wash buffer

Prepare fresh wash buffer

Store your reconstituted standards at -80°C, all other assay components 4°C.

Keep TMB substrate solution protected from light.

Precipitate in

Diluent

Precipitation and/or coagulation of components within the Diluent.

Precipitate can be removed by gently warming the

Diluent to 37ºC.

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19.

NOTES

RESOURCES

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UK, EU and ROW

Email: [email protected] | Tel: +44-(0)1223-696000

Austria

Email: [email protected] | Tel: 019-288-259

France

Email: [email protected] | Tel: 01-46-94-62-96

Germany

Email: [email protected] | Tel: 030-896-779-154

Spain

Email: [email protected] | Tel: 911-146-554

Switzerland

Email: [email protected]

Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530

US and Latin America

Email: [email protected] | Tel: 888-77-ABCAM (22226)

Canada

Email: [email protected] | Tel: 877-749-8807

China and Asia Pacific

Email: [email protected] | Tel: 108008523689 (

中國聯通

)

Japan

Email: [email protected] | Tel: +81-(0)3-6231-0940 www.abcam.com | www.abcam.cn | www.abcam.co.jp

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All information / detail is correct at time of going to print.

RESOURCES

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